Methods and systems for the rapid detection of bacteria using recombinant bacteriophage to express an indicator subunit

Abstract

Disclosed herein are methods and systems for rapid detection of microorganisms such as bacteria in a sample. A genetically modified bacteriophage is also disclosed which comprises an indicator gene encoding one subunit of an indicator protein. The specificity of the bacteriophage allows detection of a particular bacteria of interest and an indicator signal may be amplified to optimize assay sensitivity.

We claim: 
     
       1. A method for detecting a particular bacteria of interest in a sample comprising:
 incubating the sample with a recombinant indicator bacteriophage comprising an indicator gene, wherein the indicator gene encodes a first subunit of an indicator protein, thereby producing an amount of progeny phage expressing the first subunit; 
 lysing the amount of progeny phage; 
 incubating the lysed progeny phage in presence of a detection reagent, wherein the detection reagent comprises a second subunit of an indicator protein, thereby allowing the first subunit and second subunit to reconstitute to form an indicator protein complex; and 
 detecting the indicator protein complex, wherein positive detection of the indicator protein complex indicates that the particular bacteria of interest is present in the sample. 
 
     
     
       2. The method of  claim 1 , wherein the sample is a food, environmental, water, or commercial sample. 
     
     
       3. The method of  claim 1 , wherein the method detects as few as 10, 9, 8, 7, 6, 5, 4, 3, 2, or a single bacterium in a sample of a standard size for food safety industry. 
     
     
       4. The method of  claim 2 , wherein the food sample comprises meat, fish, vegetables, eggs, dairy products, dried food products, or powdered infant formula. 
     
     
       5. The method of  claim 1 , wherein the sample is first incubated in conditions favoring growth for an enrichment period of less than 24 hours, 23 hours, 22 hours, 21 hours, 20 hours, 19 hours, 18 hours, 17 hours, 16 hours, 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, or 2 hours. 
     
     
       6. The method of  claim 1 , wherein total time to results is less than 26 hours, 25 hours, 24 hours, 23 hours, 22 hours, 21 hours, 20 hours, 19 hours, 18 hours, 17 hours, 16 hours, 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, 10 hours, 9 hours 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, or 2 hours. 
     
     
       7. The method of  claim 1 , wherein ratio of signal to background generated by detecting the indicator protein complex is at least 2.0 or at least 2.5.